Abstract
A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.
Highlights
The rules of thumb for treating any infectious diseases include early yet accurate diagnosis and appropriate antibiotic treatment [1,2]
Various technologies and platforms have been introduced and applied in the development of molecular diagnostic tests for respiratory tract infections (RTIs). These include liquid-chip-based assay for viral pathogens [8], qualitative and quantitative PCR assays for individual detection of S. pneumoniae, H. influenzae, K. pneumoniae, S. aureus, P. aeruginosa, and M. tuberculosis [9,10,11,12,13], agarose gel and capillary electrophoresis-based qualitative multiplex polymerase chain reaction (mPCR) for unculturable viral and atypical bacterial pathogens [14,15], bead-based suspension array targeting 20 pathogens [16], microfluidic-based singleplex, real-time PCR using a TaqMan Array Card that can detect 32 pathogens (24 viruses, eight bacteria and two fungi) [17], and a paper/polymer hybrid microfluidic platform using loop-mediated isothermal amplification in the point-of care biochip for B. pertussis [18]
The present study describes a simplified, ready-to-use, dry-based mPCR assay targeting for H. influenzae, K. pneumoniae, M. tuberculosis, S. pneumoniae, P. aeruginosa, and S. aureus, using a panel of specific genes of the target bacteria
Summary
The rules of thumb for treating any infectious diseases include early yet accurate diagnosis and appropriate antibiotic treatment [1,2]. Failure to meet these criteria could lead to serious health issues and increase the risk of morbidity and mortality [2]. Respiratory tract infections (RTIs) have been constantly listed in the top 10 global causes of death by the World Health Organization (WHO). On top of communicable diseases in global, lower RTIs were the leading cause of death in 2019, followed by human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS), tuberculosis, and diarrheal diseases [3]. These include liquid-chip-based assay for viral pathogens [8], qualitative and quantitative PCR assays for individual detection of S. pneumoniae, H. influenzae, K. pneumoniae, S. aureus, P. aeruginosa, and M. tuberculosis [9,10,11,12,13], agarose gel and capillary electrophoresis-based qualitative mPCR for unculturable viral and atypical bacterial pathogens [14,15], bead-based suspension array targeting 20 pathogens (viruses, atypical bacteria, Acinetobacter baumannii, K. pneumoniae, S. aureus, S. pneumoniae, P. aeruginosa, and Stenotrophomonas maltophilia) [16], microfluidic-based singleplex, real-time PCR using a TaqMan Array Card that can detect 32 pathogens (24 viruses, eight bacteria and two fungi) [17], and a paper/polymer hybrid microfluidic platform using loop-mediated isothermal amplification in the point-of care biochip for B. pertussis [18]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
